Characterization of mini-protein S, a recombinant variant of protein S that lacks the sex hormone binding globulin-like domain.
Protein S is a vitamin K-dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Also, an anticoagulant role for protein S, independent of APC, has been described. Protein S has a unique C-terminal sex hormone binding globulin (SHBG)-like domain that represents about half of the molecule. To define the role of this domain in APC cofactor activity and in binding to C4b-binding protein (C4BP), we have constructed a recombinant protein S molecule of N-terminal residues 1-242 that lacks the SHBG domain (mini-protein S). A panel of monoclonal antibodies directed against the N-terminal region of protein S recognized plasma-derived protein S, wild-type recombinant protein S and mini-protein S with similar affinities, whereas a monoclonal antibody that recognizes an epitope in the SHBG domain did not detect mini-protein S. Mini-protein S did not bind to C4BP in a solid-phase binding assay, and the cofactor activity of mini-protein S was not inhibited by preincubation with C4BP. In a plasma coagulation assay, the cofactor activity of mini-protein S was lower than wild-type or plasma-derived preparations. In contrast, no difference in APC cofactor activities was observed when the preparations were tested in purified systems that monitor the APC-mediated degradation of factors Va or VIIIa. In conclusion, we constructed a protein S molecule that fails to bind C4BP and still displays cofactor activity for APC. This confirms the role of the C-terminal SHBG region in C4BP binding and demonstrates that N-terminal residues 1-242 are sufficient for the expression of APC cofactor activity in a system using purified components. In plasma, however, the C-terminal SHBG region plays a role in the expression of optimal APC cofactor activity.
Van Wijnen
M
M
Department of Haematology, G.03.647, University Hospital Utrecht, P. O. Box 85500, 3508 GA Utrecht, The Netherlands.
Stam
J G
JG
Chang
G T
GT
Meijers
J C
JC
Reitsma
P H
PH
Bertina
R M
RM
Bouma
B N
BN
D000818:Animals; D000911:Antibodies, Monoclonal; D001777:Blood Coagulation; D002352:Carrier Proteins / Q000737:chemistry*; D002460:Cell Line; D006224:Cricetinae; D015944:Factor VIIIa / Q000378:metabolism; D015943:Factor Va / Q000378:metabolism; D006801:Humans; D016167:Integrin alphaXbeta2; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism; D017293:Protein S / Q000737:chemistry*; D011994:Recombinant Proteins; D017384:Sequence Deletion; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry*; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D016167:Integrin alphaXbeta2; D011486:Protein C; D017293:Protein S; D011994:Recombinant Proteins; D012738:Sex Hormone-Binding Globulin; D015943:Factor Va; D015944:Factor VIIIa